ABSTRACT HIV-Associated Neurocognitive Disorders (HAND) and other aging-related degenerative and metabolic disorders occur with excess frequency among people living with HIV infection (PLWH), despite antiretroviral therapy and virologic suppression. Abnormal function of energy generators within cells (mitochondria) plays an important role in these complex disorders, but the basis for these abnormalities is, for the most part, unknown. While inherited mitochondrial DNA (mtDNA) mutations have been studied in relation to complex human diseases, the impact of mtDNA mutations acquired over the lifespan of an individual is only just beginning to emerge, due to the power of next-generation DNA sequencing technologies that are essential for their detection. The presence of multiple mtDNA copies within each cell in an individual (and hence many coexisting types of mtDNA) defines mitochondrial heteroplasmy, and low-frequency pathogenic, as well as common heteroplasmic mutations increase with age, even in healthy individuals, potentially crossing tissue-specific thresholds for causing disease. Some studies suggest increased mtDNA mutations and/or expansion of pre-existing heteroplasmies during HIV infection and chronic treatment. Associations between heteroplasmy and cognitive disorders and other diseases have been made outside the HIV setting. This proposal will investigate the role of mitochondrial heteroplasmy in HAND, as well as its association with known correlates of aging and HAND. A better understanding of these relationships may lead to novel risk-monitoring and treatment strategies aimed at improving mitochondrial function and reducing the risk of HAND in PLWH. We propose to leverage a unique, longitudinal aging study among PLWH, and a similarly selected HIV(-) comparison population to implement the following Specific Aims: 1) Compare changes in mtDNA heteroplasmy over a 12-year period between PLWH on antiretroviral therapy and HIV-seronegative individuals; 2a) Determine relationships between mtDNA heteroplasmy and biomarkers of cerebrospinal-fluid inflammation, oxidative stress, and systemic iron status in PLWH on suppressive antiretroviral therapy; and 2b) Determine associations of mtDNA heteroplasmy with mtDNA copy number and measures of neurocognitive performance in cross-sectional and longitudinal analyses in PLWH. For this purpose, 100 PLWH and 25 age-matched HIV-seronegative individuals will undergo deep sequencing of mtDNA and measurement of mtDNA copy number at baseline and 10-12-yr follow-up visits. Heteroplasmy associations with measured iron status and existing age- and oxidative stress biomarkers will also be performed in PLWH. The team we have assembled for this purpose has expertise in state-of-the-art next-generation sequencing methods, mtDNA heteroplasmy studies in large cohorts, and in iron-mitochondrial genomics of neuroinflammation and HAND.